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| United States Patent |
5,023,249
|
|
Kondo
, et al.
|
June 11, 1991
|
Fertility drug and method of producing the same
Abstract
A fertility drug is made from an effective component comprising ferulyl
stanol derivative represented by the following formula (I) and/or
phytosterol fatty acid ester represented by the following formula (II).
Such an effective component may be extracted from Job's tears seed,
preferably bran thereof. A synthetic method of producing ferulyl
phytostanol derivative is also disclosed.
##STR1##
| Inventors:
|
Kondo; Yoshikazu (Sendai, JP);
Suzuki; Shuetu (Tokyo, JP);
Kuboyama; Morio (Tokyo, JP)
|
| Assignee:
|
Morinaga Milk Industry Co., Ltd. (Tokyo, JP)
|
| Appl. No.:
|
433289 |
| Filed:
|
November 8, 1989 |
Foreign Application Priority Data
| Mar 05, 1985[JP] | 60-41932 |
| Mar 07, 1985[JP] | 60-43725 |
| Mar 07, 1985[JP] | 60-43726 |
| Mar 09, 1985[JP] | 60-45732 |
| Current U.S. Class: |
514/170; 514/182; 514/783; 552/653 |
| Intern'l Class: |
A01N 045/00; A01N 025/00; A61K 031/56 |
| Field of Search: |
514/170,182
552/653
|
References Cited
U.S. Patent Documents
| 4897224 | Jan., 1990 | Kondo et al. | 514/170.
|
Other References
Seiyakugaku, by Inagaki et al (1975).
Bull. of Kumamoto Local Dept. of Japan Woman Science Society, vol. 3, p.
191 (Shigemitsu (1944).
Chem. and Pharm. Bull., Japan, vol. 9, p. 43 (1961).
Chemical Abstracts, vol. 106 (1987), #84952e; Kondo et al.
Chemical Abstracts, vol. 106 (1987), #96971y; Kondo et al.
|
Primary Examiner: Friedm; Stanley J.
Assistant Examiner: Criares; Theodore J.
Attorney, Agent or Firm: Oblon, Spivak, McClelland, Maier & Neustadt
Parent Case Text
This is a continuation of application Ser. No. 06/831,853, filed on Feb.
24, 1986, now U.S. Pat. No. 4,897,224.
Claims
What we claim is:
1. A fertility drug composition, consisting essentially of, as an active
component, about 40 to 80 mg of a ferulyl stanol derivative having the
formula (I) or of a phytosterol fatty acid ester having the formula (II)
or a mixture thereof:
##STR7##
wherein R is a lower alkyl group,
##STR8##
wherein R.sub.1 is a radical of palmitic or stearic acid, and R.sub.2 is a
lower alkyl group; and a pharmaceutically-acceptable excipient.
2. The fertility drug according to claim 1, wherein said ferulyl stanol
derivative comprises transferulylstigmastanol, trans-ferulylcampestanol or
a mixture thereof.
3. The fertility drug according to claim 1, wherein said ferulyl stanol
derivative comprises a mixture of trans-ferulylstigmastanol and
transferulylcampestanol in a ratio by weight of 9:1.
4. The fertility drug according to claim 1, wherein said phytosterol fatty
acid ester comprises .beta.-sitosterol palmitic acid ester,
.beta.-sitosterol stearic acid ester, campesterol palmitic acid ester,
campesterol stearic acid ester or a combination thereof.
5. A method for producing a fertility drug, comprising adding a solvent
capable of extracting an oil-and-fat fraction to bran of Job's tears seed,
to extract an oil-and-fat fraction thereof, and isolating from said
oil-and-fat fraction a ferulyl stanol derivative having the formula (I) or
a phytosterol fatty acid ester having the formula (II) or a mixture
thereof:
##STR9##
wherein R is a lower alkyl group,
##STR10##
wherein R.sub.1 is a radical of palmitic or stearic acid, and R.sub.2 is a
lower alkyl group.
6. The method according to claim 5, which further comprises adding ethanol
to a residue of said oil-and-fat fraction to obtain an ethanol extractant,
adding ethyl acetate to said ethanol extractant to obtain an ethyl acetate
extractant, and mixing together said extractants of said first oil-and-fat
fraction and said ethyl acetate extractant.
7. The method according to claim 5, wherein said effective component is
collected by gradient elution of silica gel column chromatography with a
mixed eluent of n-hexane and ethyl acetate, said solvents being mixed in
respective amounts effective to elute said ferulyl stanol derivative or
said phytosterol fatty acid ester.
8. The method according to claim 7, wherein said ferulyl stanol derivative
represented by the formula (I) is eluted with said mixed eluent having a
ratio of n-hexane and ethyl acetate in a range of 30:1 to about 10:1.
9. The method according to claim 7, wherein said phytosterol fatty acid
ester having the formula (II) is eluted with said mixed eluent having a
ratio of n-hexane and ethyl acetate in a range of about 100:0.5 to about
100:1.
10. A ferulyl stanol derivative having the formula (I) or a phytosterol
fatty acid ester having the formula (II) or a mixture thereof:
##STR11##
wherein R is a lower alkyl group,
##STR12##
wherein R.sub.1 is a radical of palmitic or stearic acid, and R.sub.2 is a
lower alkyl group.
11. The method according to claim 5, wherein said solvent capable of
extracting an oil-and-fat fraction from bran of Job's tears seed is ethyl
acetate.
12. The fertility drug composition according to claim 1, wherein R and
R.sub.2, which are the same or different, are each methyl or ethyl.
13. The method according to claim 7, wherein R and R.sub.2, which are the
same or different, are each methyl or ethyl.
14. The ferulyl stanol derivative or the phytosterol fatty acid ester
according to claim 10, wherein R and R.sub.2, which are the same or
different, are each methyl or ethyl.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a fertility drug or an ovulation-inducing
drug and a method of producing the same, and more particularly to a
fertility drug having an effective component comprising a composition or
compositions involved in Coix lacryma-jobi var. mayuem Stapf L.
(hereinlater described simply as "Job's tears") which is extracted from
Job's tears or synthetically produced in organic reactions.
2. Prior Art Description
Typically known to the world as fertility drugs are those comprising
clomiphene and cyclohexil. These conventional fertility drugs have been
used for more than 20 years and their pharmacological effects are
recognized in clinical trials. However, it has been found in practice that
they will often cause extraordinary sex periods, resulting in such
troubles as multiple pregnancy and failure of pregnancy. They often
produce side effects. Under the circumstances, nevertheless, no other
fertility drugs have yet become available for practical purposes.
Examination and study on novel fertility drugs have been attempted. For
example, it has been known that leaves of corn, rye and wheat contain a
material which will induce ovulation of domestic rabbits (Niigata Medical
Society Bulletin; vol. 78, page 305; in 1964; Japan). Up to the present,
however, its ovulation-inducing effect on human-kind has not yet been
proved and therefore such a material is still not applicable to practical
use.
Meanwhile, it has become apparent that some pharmacological effects will
inhere in extractants from Job's tears and coix seed, a fruit of Job's
tears prepared by removing hulls and peels from Job's tears seed.
"Seiyakugaku" by Inagaki et al; published by Nankodo in 1975 in Japan;
page 162; writes as follows:
(1) The extractants of Job's tears and coix seed have diuretic effects and
therefore may be used for the remedy of tumor, beriberi, nephrolith and
cystolith and harumph.
(2) They may be used as painkillers and crampkillers.
(3) They are good for warts and roughness.
Moreover, it has now been confirmed that proteins extracted from unthreshed
powder of Job's tears seed will spur secretion of human milk (Masahiko
Shigemitsu; Bulletin of Kumamoto Local Department of Japan Women Science
Society; vol. 3, page 191; 1944). An anticancer material can be isolated
from coix seed (Chemical and Pharmaceutical Bulletin, Japan; vol. 9, page
43,; 1961). However, the ovulation-inducing effect of Job's tears and/or
the extractants thereof has not yet been known in the prior art.
SUMMARY OF THE INVENTION
It is therefore an object of the invention to provide a novel fertility
drug of a type different from the conventional one typically comprising
clomiphene and cyclohexyl, capable of inducing ovulation in
physiologically natural manner, without causing abnormal sex periods.
Another object of the invention is to provide a novel method of
synthetically producing an ovulation-inducing material, identical to that
contained in Job's tears, with high efficiency, to thereby make it
possible to manufacture on commercial basis a fertility drug having said
ovulation-inducing material as an effective component.
According to an aspect of the invention there is provided a fertility drug
having an effective component comprising oil-soluble fraction of a whole
or a part of Job's tears seed.
According to another aspect of the invention there is provided a fertility
drug having an effective component comprising ferulyl stanol derivative
and/or phytosterol fatty acid ester, contained in Job's tears. The said
fertility drug is prepared by incorporating ethyl acetate into Job's tears
bran to extract an oil-and-fat fraction from the Job's tears bran and
collecting the effective component contained in the oil-and-fat fraction.
According to still another aspect of the invention there is provided a
synthetic method of producing ferulyl phytostanol derivative comprising
the steps of acetylating ferulic acid with a mixture of pyridine and
acetic anhydride, treating the acetylated ferulic acid with thionyl
chloride to prepare an acid chloride, reacting in the presence of pyridine
the acid chloride with phytostanol to form a phytostanol compound,
dissolving the phytostanol compound into a mixture of methanol and
chloroform, and adding sodium borohydride to a resulting solution, with
stirring, to thereby deacetylate the phytostanol compound.
BRIEF DESCRIPTION OF DRAWINGS
Further objects and advantages of the present invention can be fully
understood from the following detailed description when read in
conjunction with the accompanying drawings, in which;
FIG. 1 diagrammatically illustrates the steps of the test method in Test 1
for extracting components from a whole seed of Job's tears with extracting
reagents of n-hexane, ethanol, 50% ethanol solution and water;
FIG. 2 is a graph showing relationship between dosage of a purified
extractant obtained from Job's tears seed with n-hexane and the number of
naturally produced ova and the state of ovaria;
FIGS. 3 and 4 are charts showing infra-red spectrum and nuclear magnetic
resonance spectrum of a fertility drug according to one aspect of the
invention; and
FIGS. 5 and 6 are charts showing infra-red spectrum and nuclear mabnetic
resonance spectrum of a fertility drug according to another aspect of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention according to one aspect thereof is concened with a
fertility drug or an ovulation-inducing drug having an effective component
comprising an oil-soluble fraction of a whole or a part of Job's tears
seed. The oil-soluble fraction may be extracted from powder which is
prepared by grinding the whole of the Job's tears seed. Alternatively, the
Job's tears seed is threshed and purified in a known manner to be
classified into coix seed, bran and hulls, from any of which may be
extracted the oil-soluble fraction. In view of extractability, however,
the use of bran itself or the whole seed, inclusive of the bran, is
preferred.
In extraction of the oil-soluble fraction from Job's tears seed, an
extracting solvent may be n-hexane, ethyl acetate ester or any other
suitable one. In the case of n-hexane being used, yield of the oil-soluble
fraction would be decreased but it is easier to be evaporated after
extraction, so that n-hexane is a preferred solvent in a practical sence.
The fertility drug thus prepared has an effective component of the
oil-soluble fraction extracted from Job's tears seed or a purified one
thereof. A vehicle, matching agent, diluent and any other kind of
additives may be incorporated alone or in combination, upon demand. The
fertility drug may be in the form of pellets, powder medicines, capsules,
cyrup or injections, and applied orally or externally.
For better understanding of the invention some exemplifying tests will be
given hereunder.
TEST 1
In this test, a water-soluble fraction and an oil-soluble fraction were
extracted respectively from the whole seed of Job's tears and the
ovulation-induce effects of the respective fractions were measured and
compared to each other.
The whole seed of Job's tears was ground into powder to prepare 500 g
sample. This sample powder was subjected to extraction with an extracting
reagent of 1.5 l n-hexane at a temperature of 15.degree..about.20.degree.
C. After the extraction the extracting reagent was distilled under
pressure reduced conditions to obtain about 45 g of yellow, oily substance
(extractability of about 9% by weight - the percentage is given by weight
throughout the Specification unless otherwise specified). This yellow,
oily extractant was determined as component A. Then the residue after
extraction of component A was further subjected to extraction with 1.5 l
ethanol but only a negligible amount of an extractant was obtained. The
residue was dipped into a leaching liquid of 50% ethanol solution, and the
leaching liquid was concentrated at a temperature below 40.degree. C.
under pressure reduced conditions, to thereby obtain a precipitate and
about 2 g extractant. This extractant was determined as component B. Then,
from the residue after extraction of component B was extracted with 1.5 l
water, a small amount of another extractant, which was determined as
component C. In summary, components A, B and C were extracted in such
manner as diagrammatically illustrated in FIG. 1.
These components A, B and C were qualitatively analized in a known manner
to find that component A contains glyceride and such esters as ferulic
acid ester, component B contains polyamido and component C contains amino
acid and peptide.
Next, the ovulation-inducing effect or activity of the respective
components A, B and C were tested in the following manner. More
particularly, 70 golden hamsters in the age of 5.about.8 weeks were
divided into 7 groups each having 10 golden hamsters. The fodder were
prepared by adding to the basic fodder 1% of components A, B and C, and
given orally to the group Nos. 1, 2 and 3, respectively. As the average
intake of the fodder of a golden hamster is 19 g a day, 190 mg of the
respective components were given to the respective groups. They were fed
with such fodder for 3 weeks and then slaughtered. The group Nos. 4, 5 and
6 were fed in the same manner, but after 3 weeks' feed of the fodder
containing the components only the basic fodder were given thereto for 5
weeks, and then slaughtered. Whereas, the group No. 7 was initially given
only the basic fodder for 3 weeks and then slaughtered.
As to the respective groups, the sex periods were observed and the number
of naturally produced ova was counted before they were slaughtered, and
after slaughtered the weight and the state of the ovary were observed by
means of dissection. The results of these tests are shown in the following
Table I.
TABLE I
__________________________________________________________________________
test sample
comparison of
number of
dissection test
group
component
sex period natural
weight of
state of
No. added sex period
remarks
ovulation
ovary (mg)
ovary
__________________________________________________________________________
1 A 4 days
regular
17 .+-. 2
19.48 .+-. 2.35
(++)
2 B 4 days
regular
12 .+-. 2
18.01 .+-. 0.34
(.+-.)
3 C 4 days
regular
12 .+-. 2
17.93 .+-. 0.23
(.+-.)
4 A 4 days
regular
17 .+-. 3
19.59 .+-. 0.23
(++)
5 B 4 days
regular
12 .+-. 2
18.20 .+-. 0.26
(+)
6 C 4 days
regular
12 .+-. 2
17.83 .+-. 0.56
(.+-.)
7 -- 4 days
regular
12 .+-. 2
18.03 .+-. 0.15
(.+-.)
__________________________________________________________________________
Notes:
(1) Figures in the column of Sex Period show the average.
(2) In the column of State of Ovary the marks (.+-.), (+) and (++) mean
relative comparison in the number of corpora lutea found in cutouts of th
ovary.
(3) The mark () identified in the column of Natural Ovulation means that
there could be found a significance with respect to other groups at a
significance level of 1%.
As it appears from the results shown in Table I, the component A extracted
from the whole seed of Job's tears with n-hexane will promote natural
ovulation without disturbing the sex periods. As a result of organic
dissection, the effective component contained in the component A was
proved to act on the sex center so as to promote formation of corpora
lutea.
Meanwhile, Table I also shows that this effective component is contained
only in the oil-soluble fraction of the Job's tears seed and is scarcely
present in the extractants obtained with ethanol, 50% ethanol solution and
water.
TEST 2
This test was carried out to find the optimal dosage of the
ovulation-inducing effective component contained in the component A
extracted in Test 1.
First, the component A was dipped into leaching liquids of ethanol, 50%
ethanol solution and water, step by step, and then the leaching liquids
were removed. Then the residue was purified by column chromatography with
n-hexane. Thus, a purified extractant was obtained in a yield of 90% of
the component A.
This extractant was dissoved in 0.2 ml. soybeam oil in quantities varied
between 95.about.380 mg to prepare three fodder containing different
quantities of the extractant, which were orally applied by injector to
three groups each having 10 golden hamsters, once a day, for 3 weeks.
Thereafter, the test animals were slaughtered. These groups were put into
the tests for the natural ovulation and the ovary weight in the same
manner as in Test 1, upon which matural relation of the dosage of the
extractant per body weight of a golden hamster was determined with these
parameters. The test results are shown in FIG. 2.
As shown, the optimal dosage per body weight of a golden hamster was found
to be in a range of 0.76.about.1.4 mg/g a day. Accordingly, provided the
body weight of a grownup is 60 kg, 4.6.about.8.4 g/day of the purified
n-hexane extractant was determined to be an optimal range when applied to
an adult. The medication term will be dependent upon the condition of a
patient and varied case by case.
TEST 3
In this test, extractability of the effective component obtained
respectively from the whole seed, coix seed, bran and hull of Job's tears
was measured and compared to each other.
Respective 500 g powder were prepared by grinding the coix seed, bran and
hull of Job's tears seed, and extractants corresponding to component A
were extracted in the same manner as in Test 1. Quantities of the
extractants thus obtained and extractability in the respective cases are
shown below in Table II. Data for the whole seed in Table II are the
reproduction of those in Table I. It will be obvious from Table II that
the greatest extractability was gained in the case of bran, leading to the
fact that effective component is in substance contained in the bran of
Job's tears seed. Thus, the effective component can be extracted with
great efficiency from the bran alone or the whole seed including the bran.
TABLE II
______________________________________
test sample
amount of extractant (g)
extractability (%)
______________________________________
whole seed
45.0 9.0
coix seed 27.0 5.4
bran 80.0 16.0
hull 0.6 0.12
______________________________________
Note: The extractability is calculated by the equation of amount of
extractant (g)/sample powder (500 g) .times. 100
The inventors have made thorough investigation of the effective
ovulation-inducing material contained in Job's tears seed and found in the
end that ferulyl stanol derivatives expressed by the following formula (I)
and/or phytosterol fatty acid ester expressed by the following formula
(II) has significant effect and activity on induction of natural
ovulation. Based on this fact the inventors have come to a successful end
that a novel fertility drug having an effective component comprising
ferulyl stanol derivatives and/or phytosterol fatty acid ester may be
prepared.
##STR2##
Stanol ferulic acid derivatives represented by the formula (I) may be, for
example, trans-ferulyl stigmastanol, trans-ferulyl campestanol or a
mixture thereof.
Phytosterol fatty acid ester represented by the formula (II) may be, for
example, .beta.-sitosterol palmitic acid ester, .beta.-sitosterol stearic
acid ester, campesterol palmitic acid ester, campesterol stearic acid
ester or any one of the combinations thereof.
A process for producing the above fertility drug is described in detail
hereunder.
First, a Job's tears seed is threshed and purified in a known manner to be
separated into coix seed, bran and hull. To 1 part of the bran is
incorporated 3.about.5 parts (by weight; the part is given by weight
throughout the Specification unless otherwise specified) of ethyl acetate,
and the resultant is agitated at 15.degree..about.20.degree. C. for
5.about.10 hours, thereby extracting an oil-and-fat fraction. The extract
is then filtered to remove an insoluble fraction.
To 1 part of the residue is incorporated 3.about.5 parts of ethanol, and
the solution is agitated at 15.degree..about.20.degree. C. for 5.about.10
hours. The resultant extract is then filtered to remove an insoluble
fraction. The ethanol-soluble fraction thus obtained is distilled to
remove ethanol, thereby obtaining an ethanol extractant. To 1 part of the
ethanol extractant is added 3.about.5 parts of ethyl acetate, and the
solution is agitated at 15.degree..about.20.degree. C. for 5.about.10
hours, to obtain an ethyl acetate-soluble fraction.
The oil-and-fat fraction and the ethyl acetate-soluble fraction are mixed
together and ethyl acetate is separated from the mixture. The resultant is
supplied to a column charged with silica gel to effect gradient elution
with a mixed eluent of n-hexane and ethyl acetate, whereby fractions
eluted with the mixed eluents of n-hexane and ethyl acetate having mixing
ratios of 30:1.about.10:1 and/or 100:0.5.about.100:1 are collected. The
fractions thus collected are mixed together and the eluents are removed
from the mixture, thereby obtaining ovulation-inducing materials. The
fractions eluted with the eluent of 30:1.about.10:1 mixture of n-hexane
and ethyl acetate will contain ferulyl stanol derivatives and the
fractions eluted with the eluent having the mixing ratio of
100:0.5.about.100:1 will contain phytosterol fatty acid ester. Effective
materials will be contained in the respective eluates obtained with the
eluents having different ranges of mixing ratio and may be used alone or
in a mixed state to manufacture the fertility drug according to the
invention. The drug may be a pellet, a powder medicine, a capsule, an
injection or in any other form, and applied orally or externally. The
dosage of the drug will vary depending upon the condition of a patient and
the way of application, but in most cases 40.about.80 mg once a day will
be effective when orally applied to an adult.
The fertility drug of the invention having an effective component
comprising ferulyl stanol derivative and/or phytosterol fatty acid ester
will be further described by way of exemplifying tests.
TEST 4
This test was carried out to observe physical and chemical characteristics
of ferulyl stanol derivative. The test sample was X component prepared in
the same manner as in Example 3, described later.
This test sample was analized by thin-layer chromatography to detect a
single spot (hereinlater defined as material X) which manifested a
blue-green color with sulfuric acid and was distinguishable in ultraviolet
rays.
The material X was purified by alumina column chromatography and isolated
as colorless, needle-like crystals having melting point of 156.degree. C.
The material X showed positive adsorpotion in the Gibbs' adsorption test
and had a M.sup.+ peak at m/e 592 in the mass spectrometry. In the
infra-red spectrophotometry, as shown in FIG. 3, it showed adsorption
signals based on hydroxyl group at 3120.about.3500 cm.sup.-1, conjugated
carboxyl group at 1710 cm.sup.-1, double-bond of
.alpha.,.beta.-unsaturated carbonyl group at 1640 cm.sup.-1, C--C
stretching vibration of benzene ring at 1600 and 1510 cm.sup.-1, and
out-of-plane deformation vibration of benzene ring at 840 cm.sup.-1,
respectively. In the nuclear magnetic resonance spectrometry (CDCl.sub.3),
as shown by the chart of FIG. 4, it manifested a signal pattern peculiar
to phytosterol at 0.62.about.2.0 ppm. Further, signals were found based on
methoxy group at 3.88 ppm, and the AB type spin-spincoupling (J=16Hz) of
hydrogen in the double-bond of .alpha.,.beta.-unsaturated carbonyl group
of cinnamic acid derivative and and the ABM type spin-spincoupling of
hydrogen in the tri-substituted benzene ring thereof was found at 6.22 ppm
and 7.55 ppm, respectively. The singlet of 1H at 5.95 ppm was disappeared
by adding D.sub.2 O. According to these results, the material X was
determined to have hydroxyl group of a phenol.
The material X was subjected to alkalic hydrolysis to be separated into an
acid and a neutral fractions, to both of which were applied chemical
analysis to prove that only ferulic acid was isolated from the acid
fraction (which was identified in comparison with specimen). The neutral
fraction was silylated in a known manner and then quantitatively analyzed
by gas chromatography, whereby it was identified as a mixture of
stigmastanol and campestanol in a ratio of 9:1.
Further, the material X was acetylated with a mixture of pyridine and
acetic acid to form mono-acetal in the form of colorless, platy crystals,
having melting point of 155.degree..about.156.degree. C. According to
nuclear magnetic resonance spectrometry (CDCl.sub.3), there could be seen
signals based on methyl and methylene radicals of phytosterol at
0.6.about.2.0 ppm, and a signal based on acetyl group at 2.32 ppm. At 3.84
ppm, a signal of singlet of 3H was found based on methyl of methoxy group.
The AB type spin-spincoupling of olefin hydrogen in
.alpha.,.beta.-unsaturated carboxyl group was noted at 6.32 ppm and 7.60
ppm, and the ABM type spin-spincoupling of hydrogen on the tri-substituted
benzene ring appeared at 7.07 ppm.
Consequently, the material X was identified as a mixture of transferulyl
stigmastanol and trans-ferulyl campestanol in a ratio of 9:1. By the
results of analyses including its melting point, the transferulyl
stigmastanol was found to be the same material as
dihydro-.beta.-sitosterol ferulic acid ester which is isolated from a corn
embryo bud oil (Tamura et al; "Nippon Kagaku Zasshi (Japan Chemical
Magazine)"; vol. 79, page 1011; in 1958).
TEST 5
This test was carried out to reveal physical and chemical characteristics
of phytosterol fatty acid ester. Y component, prepared in the same manner
as in Example 3, described later, was used as a test sample.
This sample was analized by thin-layer chromatography in a known manner to
isolate a material of colorless, needle-like crystals, having a melting
point of 64.degree..about.65.degree. C., which showed a spot manifesting a
blue-green color with sulfuric acid and being distinguishable in
ultraviolet rays. According to mass spectrometry, a peak based on M.sup.+
was found at m/e 680. A peak based on calbonyl group was observed at 1740
cm.sup.-1 in infra-red spectrophotometry, as shown in FIG. 5. In nuclear
magnetic resonance spectrometry (CDCl.sub.3), as shown by the chart in
FIG. 6, signals were noted based on methyl and methylene protons of
phytosterol at 0.6.about.2.0 ppm, and --(CH.sub.2)-- of long chain fatty
acid residue at 1.28 ppm. A signal of triplet based on methylene group
adjacent carbonyl groups, a signal of multiplet (12 Hz in half width)
based on hydrogen of C.sub.3 phase and a signal of multiplet based on
binyl hydrogen were found at 2.25 ppm, 4.6 ppm and 5.35 ppm, respectively.
Then, the material was subjected to alkalic hydrolysis to be classified
into an acid and a neutral fractions. The neutral fraction was proved to
contain sterol, which comprised .beta.-sitosterol and campesterol. The
acid fraction was a mixture of higher fatty acid comprising in substance
stearic acid and palmitic acid, which was determined by means of
methylation and gas chromatography.
In conclusion, the said Y component was identified as a mixture of long
chain fatty acid esters of phytosterol. A part of this material was
identical to .beta.-sitosterol derivative mentioned by Kuksis et al in
"Journal of Organic Chemistry", vol. 0.25, page 1209; in 1960.
TEST 6
In this test yield of stanol ferulic acid derivative and phytosterol fatty
acid ester from the whole seed, coix seed, bran and hulls of Job's tears
was compared to each other.
The whole seed of Job's tears was ground in a known manner, threshed and
purified, and thus separated into hulls, bran and coix seed. Among 100
parts of the whole seed, 33 parts of the hulls, 15 parts of the bran and
52 parts of the coix seed were obtained. To these four starting materials
were respectively added n-hexane and ethyl acetate each in a triple
quantity thereof, and the solution was agitated at
15.degree..about.20.degree. C. for 5 hours, to thereby effect extraction.
After the respective extracting solvents had been removed, oily components
were obtained. The weight ratios of the respective oily components with
the starting materials are shown in the following Table III, which shows
the fact that the effective components comprising ferulyl stanol
derivative and phytosterol fatty acid ester are both included in the bran
of Job's tears seed.
TABLE III
__________________________________________________________________________
oily component
oily component
yield per 1 g oily component (g)
starting
extracted with
extracted with
ferulyl stanol
phytosterol
material
n-hexane
ethyl acetate
derivative
fatty acid ester
__________________________________________________________________________
whole
9.3 12.2 0.0011 0.0201
hull 0.12 0.18 little little
bran 16.0 25.1 0.0011 0.0145
coix seed
5.4 7.3 little little
__________________________________________________________________________
TEST 7
This test was carried out to determine fractions containing the effective
components comprising ferulyl stanol derivative and phytosterol fatty acid
ester, by means of silical gel chromatography.
(1) Preparation of Test Samples
According to the method described later in Example 3, 1,250 g of an oily
fraction was extracted from 5 kg of Job's tears seed bran with ethyl
acetate and ethanol.
300 g of oily fraction was subjected to column chromatography using 5 kg of
silica gel, wherein the eluent was at first n-hexane and then mixtures of
n-hexane and ethyl acetate in mixing ratios being varied to gradually
increase the proportion of ethyl acetate. The eluates were 4.352 g of F-I
component eluted with a mixed eluent of n-hexane and ethyl acetate in a
mixing ratio of 100:1, 283 g of F-II component with an eluent of a 20:1
mixture and 8.756 g of F-III component with ethyl acetate alone. F-I
component was purified by column chromatography using 250 g silica geland
a mixed eluent of n-hexane and ethyl acetate in a ratio of 100:1. F-II
component was further subjected to alumina column chromatography and
eluted with a 20:1 mixture of n-hexane and ethyl acetate to obtain 128 mg
of F-II-1 component, 1.284 g of F-II-2 component, 210 mg of F-II-3
component, 210 mg of F-II-4 component and 55 mg of F-II-5 component. Thus,
seven specimens were prepared.
(2) Test Method for Physiological Activities
These seven specimens were dissolved into 0.2ml of a soy bean oil to
prepare various oil solution, which were orally given once a day to
respective groups each having 10 golden hamsters in the age of 5.about.8
weeks. The amounts of the components respectively contained in such oil
solutions were made different to be 0.2 mg and 0.5mg. During the
administration for 3 weeks with such oil solutions, the sex periods and
the number of naturally produced ova were observed, the results of which
were compared with those of reference group which had been treated with
0.2 mg of soy beam oil containing no sample component.
(3) Results
The results with respect to the natural ovulation are shown in Table IV.
TABLE IV
______________________________________
component amount of component added
added 0.2 mg 0.5 mg
______________________________________
(reference) 12 12
F-I 18 15
F-II-1 12 10
F-II-2 11 10
F-II-3 12 12
F-II-4 18 13
F-II-5 12 11
F-III 11 12
______________________________________
The sex periods were 4 days for all groups including the reference and
there could be found no disturbing effect on the sex period. It will be
quite obvious that F-I and F-II-4 components have significant
ovulation-induce effect. These components were identified in a known
manner to determine that F-I component was phytosterol fatty acid ester
and F-II-4 component was ferulyl stanol derivative.
TEST 8
This test was performed to determine effective dosage of stanol ferulic
acid derivative, an effective ovulation-inducing component of the
fertility drug according to the invention.
(1) Preparation of Test Sample
X component was prepared in the same manner as in Example 3, described
later, and used as a test sample. X component was a 9:1 mixture of
trans-ferulylstigmastanol and trans-ferulylcampestanol.
(2) Test Method
The test was carried out in the same manner as in Test 7, except the
amounts of the component added in the soy bean oil were 0.1 mg, 0.2 mg and
1.0 mg once a day, respectively.
(3) Results
(3-1) Sex Period
TABLE V
______________________________________
amount of component added
sex period (day)
______________________________________
(reference) 4
0.1 mg 4
0.2 mg 4
1.0 mg 4
______________________________________
As shown from Table V, each group showed regular sex period.
(3-2) Number of Natural Ovulation
TABLE VI
______________________________________
amount of component
number of natural
added ovulation
______________________________________
(reference) 11 .+-. 1
0.1 mg 16 .+-. 1
0.2 mg 18 .+-. 2
1.0 mg 13 .+-. 2
______________________________________
Note: The mark shows a significance at a significance level of 1%.
As shown from Table VI, the groups to which 0.1 mg and 0.2 mg of the
effective components were given show significance with P<0.01 with respect
to the reference group.
As the average body weight of a hamster used in this test was 150 g and the
body weight of an adult is supposed to be 60 kg, the effective dosage to
an adult is proved to be 40.about.80 mg once a day.
TEST 9
This test was perfomed to determine effective dosage of phytosterol fatty
acid ester, another effective ovulation-inducing component of the
feritility drug according to the invention.
(1) Preparation of Test Sample
Y component was prepared in the same manner as in Example 3, described
later, and used as a test sample.
(2) Test Method
The test was carried out in the same manner as in Test 7, except the
amounts of the component added in the soy bean oil were 0.1 mg, 0.2 mg and
1.0 mg once a day, respevtively.
(3) Results
(3-1) Sex Period
TABLE VII
______________________________________
amount of component added
sex period (day)
______________________________________
(reference) 4
0.1 mg 4
0.2 mg 4
1.0 mg 4
______________________________________
It will be quite obvious from Table VII that each group showed regular sex
period.
(3-2) Number of Natural Ovulation
TABLE VIII
______________________________________
amount of component
number of natural
added ovulation
______________________________________
(reference) 12 .+-. 2
0.1 mg 16 .+-. 1
0.2 mg 18 .+-. 2
1.0 mg 15 .+-. 1
______________________________________
Note: The mark shows a significance at a significance level of 1%.
As shown from Table VIII, the groups to which 0.1 mg and 0.2 mg of the
effective components were given show significance with P<0.01 with respect
to the reference group.
As the average body weight of a hamster used in this test was 150 g and the
body weight of an adult is supposed to be 60 kg, the effective dosage to
an adult is proved to be 40.about.80 mg once a day.
Ferulyl phytostanol derivative, one of the effective components having
ovulation-inducing effect, may be prepared not only by extraction from
Job's tears seed as described before, but also by synthetic method. Such a
synthetic method of production of ferulyl phytostanol derivative is
characterized by successive steps of acetylating ferulic acid with a
mixture of pyridine and acetic anhydride, treating the acetylated ferulic
acid with thionyl chloride to form an acid chloride, reacting in the
presence of pyridine the acid chloride and phytostanol to prepare
phytostanol compound, dissolving the phytostanol compound into a mixture
of methanol and chloroform, and adding sodium boron hydride to a resulting
solution, with stirring, to thereby deacetylate the phytostanol compound.
The respective steps will be described in detail as follows.
(A) Acetylation
Ferulic acid (4-hydroxy-3-methoxycinnamic acid) is dissolved into a liquid
mixture of pyridine and acetic anhydride (mixing ratio of 3:1) in a
concentration of 10.about.15 wt. %. After reflux for 4.about.5 hours the
solvent is distilled to prepare acetylated ferulic acid.
##STR3##
(B) Formation of Acid Chloride
The acetylated ferulic acid is dissolved into chloroform anhydride in a
concentration of 10.about.15 wt. %. Thionyl chloride (SOCl.sub.2) is added
to the solution in a proportion of 1.5 moles per 1 g acetylated ferulic
acid to effect the reaction thereof at 20.degree..about.30.degree. C. for
5.about.6 hours. Then, the solvent is distilled in pressure reduced
conditions to prepare acid chloride.
##STR4##
(C) Reaction with Phytostanol
The acid chloride thus prepared is dissolved into pyridine in a
concentration of 10.about.15 wt. % to prepare a first solution, and an
equivalent of phytostanol is dissolved into pyridine to prepare a second
solution. The first solution is dropped into the second solution in an
ice-cooled condition to effect the reaction at 20.degree..about.30.degree.
C. for 2.about.3 hours, and the solvent is distilled and removed.
##STR5##
(D) Deacetylation
The reactant thus obtained in the step (C) is dissolved into a liquid
mixture of chloroform and methyl alcohol (mixing ratio of 1:1) in a
concentration of 3.about.5 wt. %. While this solution is being agitated in
an ice-cooled condition, sodium borohydride in a quantity of double
equivalent is added thereto by degree. After the foaming has ceased the
solution is allowed to stand at 20.degree..about.25.degree. C. for
1.about.1.5 hours, a small quantity of water is added and then the solvent
is evaporated in pressure reduced conditions. Thus, ferulyl phytostanol
derivative is prepared.
##STR6##
Ferulyl phytostanol derivative thus prepared by the successive steps (A) to
(D) may be further purified by silica gel column chromatography and then
recrystalized with methyl alcohol, thereby producing the same in a pure
state. Alternatively, the purification may be carried out each time the
intermediate product is formed in each step.
The following are exemplifying tests for the method of producing ferulyl
phytostanol derivative according to the invention.
TEST 10
This test was carried out to examine physical and chemical characteristics
of the intermediate product formed by the acetylation step (A).
(1) Preparation of Test Sample
Test sample was prepared in the same manner as the acetylation step in
Example 6, described later. The acetylated product was subjected to silica
gel column chromatography whereby a fraction eluted with acetone was
obtained, from which acetone was evaporated to prepare a purified test
sample.
(2) Test Method
The test sample was put into tests for melting point, mass spectrometry,
infra-red spectrophotometry (KBr method) and nuclear magnetic resonance
spectrometry (CD.sub.3 OD+CDCl.sub.3) in known manner.
(3) Results
The test results were as follows.
mp. 195.degree..about.196.degree. C., FD-MS m/e; 236 (M.sup.+) IR.nu. (KBr)
Cm.sup.-1 ; 3500 (COOH), 1760,1690 (>C.dbd.O), 1630 (--COOH.dbd.CH--), NMR
(CD.sub.3 OD+CDCl.sub.3), ppm: 2.32 (3H, S, COCH.sub.3), 3.82 (3H, S,
OCH.sub.3), 6.32 (1H, d, J.dbd.16Hz, --COCH.dbd.CH--), 7.07 (3H, m,
aromatic ring), 7.60 (1H, d, J.dbd.16Hz, --COCH.dbd.CH--).
Consequently, the test sample was identified as 4-acetylferulic acid
(4-acetoxy-3-methoxycinnamic acid).
TEST 11
This test was carried out to observe physical and chemical characteristics
of the intermediate product formed by the reaction of acetylated ferulic
acid chloride and stigmastanol.
(1) Preparation of Test Sample
Test sample was prepared in the same manner as the step of formation of
acid chloride in Example 6, described later. The acid chloride compound
thus prepared was subjected to silica gel column chromatography with a
mixed eluent of n-hexane and ethyl acetate ester (mixing ratio of 20:1).
The eluate was recrystalized with methyl alcohol to prepare a purified
test sample.
(2) Test Method
The tests applied to the sample were the same as in Test 10. The elementary
analysis was carried out in a known manner.
(3) Results
A melting point was 156.degree. C. The results of the elementary analysis
were C: 77.80 and H: 9.89, which are in conformaty to the calculated
values of C: 77.60 and H: 9.78 according to the molecular formula of
C.sub.41 H.sub.62 O.sub.5 of 4-acetylferulylstigmastanol.
The results of mass spectrometry, infra-red spectrophotometry and nuclear
magnetic resonance spectrometry were as follows.
FD-MS m/e; 634 (M.sup.+), IR.nu. (KBr): 1770,1710 (>C.dbd.O), 1640
(--COOH.dbd.CH--), 1600,1510 (benzene ring), 1180,1080 (--C--O--C--), NMR
(CDCl.sub.3), ppm: 0.6.about.2.0 (--CH.sub.3, --CH.sub.2 --), 2.32 (3H, S,
COCH.sub.3), 3.84 (3H, S, OCH.sub.3), 4.70 (1H, m, --O--CH<), 6.32 (1H, d,
J.dbd.16Hz, --COCH.dbd.CH--), 7.07 (3H, m, aromatic ring), 7.60 (1H, d,
J.dbd.16Hz, --COCH.dbd.CH--).
In accordance with these results the test sample was identified as
4-acetylferulylstigmastanol.
TEST 12
This test was performed to test physical and chemical characteristics of
the final product prepared by the method of the invention.
(1) Preparation of Test Sample
Test Sample was prepared and purified by the same method as in Example 6.
(2) Test Method
The same tests were applied as in Test 11.
(3) Results
A melting point was 152.degree. C. The results of the elementary analysis
were C: 79.17 and H: 9.86, which are in good agreement with those of C:
79.05 and H: 10.14 calculated according to the molecular formula of
C.sub.39 H.sub.60 O.sub.4.
The results of mass spectrometry, infra-red spectrophotometry and nuclear
magnetic resonance spectrometry were as follows.
FD-MS m/e; 592 (M.sup.+), IR.nu. (KBr); Cm.sup.-1 :3200-3500 (--OH), 1710
(--CO), 1640 (--COOH.dbd.CH--), 1600,1520 (benzene ring), 1180,1080
(--C--O--C--), NMR (CDCl.sub.3), ppm: 0.6.about.2.0 (--CH.sub.3, CH.sub.2
of sterol<), 3.88 (3H, S, --OCH.sub.3), 4.72 (1H, m, --O--CH<), 5.96 (1H,
S, --OH), 6.22 (1H, d, J=16Hz, --COCH.dbd.CH--), 6.98 (3H, m, aromatic
ring), 7.55 (1H, d, J=16Hz, --COCH.dbd.CH--).
In conclusion, the final product was identified as trans-ferulyl
stigmastanol.
For better understanding of the invention some preferred examples thereof
will be given hereunder.
EXAMPLE 1
5 kg powder of Job's tears prepared by grinding the whole seed thereof was
subjected to extraction with 15 l n-hexane at a temperature of 20.degree.
C. and the solvent was evaporated at a temperature below 40.degree. C.
under reduced pressure, thereby obtaining about 450 g of a yellow, oily
material at extractability of about 9%. This extractant was then purified
by silica gel chromatography with n-hexane. The purified extractant was
added to basic feed to prepare fodder which was given to a group having 10
golden hamsters in the age of 5.about.8 weeks. The quantities of the
fodder given to a golden hamster and the purified n-hexane extractant
contained therein were determined 19 g and 171 mg a day, respectively.
While the test animals were fed for 3 weeks the sex periods were observed;
and thereafter they were slaughtered to measure the number of naturally
produced ova and observe the state of the ovaria. For reference, an
equivalent quantity of the basic fodder not containing the purified
n-hexane extractant, was given to another group also having ten golden
hamsters, to which the same tests were applied.
The test results are shown in Table IX, from which it is confirmed that the
oil-soluble fraction of the whole seed of Job's tears has a favorable
effect on natural ovulation and formation of corpora lutea, without
disturbing sex periods.
TABLE IX
__________________________________________________________________________
comparison of
number of
dissection test
sex period natural
weight of
state of
test group
sex period
remarks
ovulation
ovary (mg)
ovary
__________________________________________________________________________
this invention
4.5 days
regular
16 .+-. 2
19.25 .+-. 1.66
(++)
reference
4 days
regular
12 .+-. 2
18.08 .+-. 0.16
(.+-.)
__________________________________________________________________________
Note: The representation of sex period and the marks identified above sho
the same meanings as in Table I.
EXAMPLE 2
In Example 1 was used the whole seed of Job's tears but in this example 500
g bran of Job's tears was used, and by extraction with 1.5 l n-hexane at
20.degree. C. was obtained about 80 g of a yellow oily material at
extractability of about 16%. This material was purified by silica gel
chromatography with n-hexane and the solvent was evaporated at 40.degree.
C. under reduced pressure, thus preparing about 72 g of a purified
extractant. This purified extractant was given to a group of ten golden
hamsters in a proportion of 1.5 mg a day per 1 g of the body weight of a
golden hamster, and the same tests were put into practice as in Example 1,
results of which are shown in Table X. As shown, the oil-soluble fraction
of the bran of Job's tears seed will promote natural ovulation without
disturbing the sex period and bring a significant effect on formation of
corpora lutea.
TABLE X
__________________________________________________________________________
comparison of
number of
dissection test
sex period natural
weight of
state of
test group
sex period
remarks
ovulation
ovary (mg)
ovary
__________________________________________________________________________
this invention
4 days
regular
18 .+-. 2
19.48 .+-. 1.51
(++)
reference
4 days
regular
12 .+-. 2
17.92 .+-. 0.23
(.+-.)
__________________________________________________________________________
Note: The representation of sex period and the marks identified above sho
the same meanings as in Table I.
EXAMPLE 3
50 kg of Job's tears seed was threshed and purified in known manner to
prepare about 6.5 kg bran, among which 5 kg bran was subjected to
extraction with 5 kg ethyl acetate at 20.degree. C. for 5 hours while
being agitated. This extraction procedure was repeated three times to
obtain a first extractant. To 4 kg of the residue was added 10 kg ethanol,
and the reaction solution was agitated at 20.degree. C. for 5 hours. This
second extraction procedure was repeated twice and then the collected
extracs were filtered to remove insoluble fractions. From the
ethanol-soluble fraction thus obtained was evaporated ethanol in a known
manner. To 120 g of the resulting component was added 360 g ethyl acetate
and the solution was agitated at 20.degree. C. for 5 hours, to extract an
ethyl acetate soluble fraction, a second extractant.
The first and second extractants were mixed together. Ethyl acetate was
evaporated and removed from the extractant mixture to obtain 1,310 g of an
oil-and-fat fraction.
A first portion of 500 g of the oil-and-fat fraction was subjected to
column chromatography charged with 8 kg silica gel, whereby 450 g eluate
was obtained with a mixed eluent of n-hexane and ethyl acetate (mixing
ratio of 20:1). This eluate was further introduced to alumina column
chromatography (800 g) whereby elution was effected with a mixed eluent of
n-hexane and ethyl acetate (mixing ratio of 20:1) and about 540 mg of a
fraction showing a spot in ultraviolet rays was obtained by a fraction
collector. This component was subjected to reverse phase high performance
liquid chromatography utilizing Rp-18 with a mixed eluent of ethyl acetate
and methanol (mixing ratio of 5:3), to obtain about 450 mg of a component
having a U V absorption. This component was further subjected to column
chromatography using 50 g of silica gel with a mixed eluent of n-hexane
and ethyl acetate (mixing ratio of 20:1), to elute about 330 mg of a
component (X component) having a U V absorption. X component was put into
the same tests as in Test 7 so that it was proved to have
ovulation-inducing effect.
On the other hand, another portion of 500 g of the oil-and-fat fraction was
subjected to column chromatography using 8 kg silica gel, with a mixed
eluent of n-hexane and ethyl acetate (mixing ratio of 100:0.5), then an
eluate (Y component) was obtained in a quantity of about 6.87 g. Y
component was also submitted to the same tests as in Test 7 and its
ovulation-inducing effect was observed.
EXAMPLE 4
To 3 kg of Job's tears bran was added 3 kg ethyl acetate and the solution
was agitated at 17.degree. C. for 8 hours for effecting extraction. This
extraction procedure was repeated four times and extractants obtained in
respective extraction were combined. Thus, an ethyl acetate extractant was
prepared. To 2.2 kg of the residue was added 8 kg ethanol and the mixture
was agitated at 17.degree. C. for 8 hours. The extract thus obtained was
filtered to remove insoluble components. By evaporating ethanol from the
ethanol-soluble fraction in a known manner, an extractant with ethanol was
obtained in a quantity of 65 g. To this extractant was added 260 g ethyl
acetate, and the solution was agitated at 17.degree. C. for 8 hours, to
extract an ethyl acetate soluble extractant. These two ethyl acetate
extractants were combined, from which ethyl acetate was evaporated to
obtain 790 g of an oil-and-fat fraction.
A first portion of 300 g of the oil-and-fat fraction was subjected to
column chromatography using 5 kg silica gel, with a mixed eluent of
n-hexane and ethyl acetate (a mixing ratio of 30:1), to obtain an eluate
in a quantity of 269 g. This eluate was subjected to alumina column
chromatography (500 g), thereby obtaining 330 mg of a fraction having a
spot in ultraviolet rays. This fraction was subjected to reverse phase
high performance liquid chromatography utilizing Rp-18, thereby eluting
with a mixed eluent of ethyl acetate and methanol (a mixing ratio of 5:3)
to obtain about 248 mg of a component having a U V absorption. This
component was further subjected to column chromatography using 30 g silica
gel with a mixed eluent of n-hexane and ethyl acetate (a mixing ratio of
30:1) to obtain about 190 mg of a fraction having a U V absorption. This
fraction was submitted to tests in the same manner as in Test 7 so that it
was proved to have ovulationinducing effect.
On the other hand, the other portion of 300 g of the oil-and-fat fraction
was subjected to column chromatography using 5 kg silica gel with a mixed
eluent of n-hexane and ethyl acetate (a mixing ratio of 100:1) to obtain
4.352 g of an eluate. This eluate was tested in the same manner as in Test
7 to find its significant ovulation-inducing effect.
EXAMPLE 5
The procedure in Example 1 was repeated several times to obtain 40 g of X
and Y components, respectively, to which 150 g of middle-chain fatty acid
triglycerol on the market was added to make 1,000 soft capsule medicines
therefrom, respectively. A soft capsule medicine made from X component
contained 40 mg of the effective component (that is, a 9:1 mixture of
trans-ferulylstigmastanol and trans-ferulylcampestanol) and a soft capsule
medicine made from Y component contained 80 mg of the effective component
(that is, phytosterol long-chain fatty acid esters in a mixed state).
EXAMPLE 6
According to the following steps trans-ferulyl stigmastanol was
synthetically manufactured.
(1) Acetylation
1.0 g of ferulic acid (Tokyo Kasei Kougyo K. K., corresponding to 5.15 m
moles) was dissolved into 8 ml of a liquid mixture of pyridine and acetic
anhydride in a mixing ratio of 3:1, and after reflux for 4 hours the
solvent was evaporated.
(2) Formation of Acid Chloride
1.0 g of the acetylated ferulic acid (4.24 m mol) was dissolved into 10 ml
chloroform anhydride and 0.46 ml (6.36 m moles) of thionyl chloride (Wako
Jun'yaku K. K.) was added to the solution, to effect the reaction thereof
at 25.degree. C. for 5 hours, and then the solvent was evaporated under
reduced pressure.
(3) Reaction with Phytostanol
The acid chloride compound thus obtained was dissolved into 10 ml pyridine
anhydride and the resultant was dropped in an ice-cooled condition into 10
ml of another pyridine solution prepared by dissolving thereto 1.77 g
(4.24 m moles) of stigmastanol (Aldrich Chemical Co.,). After the solution
had been allowed to stand at room temperature for 2 hours, the solvent was
evaporated. The remaining residue was subjected to a silica gel column,
and the column was eluted with a mixed eluent of n-hexane and ethyl
acetate (a mixing ratio of 20:1). Thus, a reactant was obtained in a
quantity of 1.65 g.
(4) Deacetylation and Purification
1.6 g of the reactant was dissolved into 48 ml of a liquid mixture of
chloroform and methyl alcohol (a mixing ratio of 1:1), and 480 mg (12.7 m
moles) of sodium borohydride (Wako Jun'yaku K. K.) was added thereto
little by little. After the foaming had ceased the reaction mixture was
allowed to stand for 1 hour, and then 1 ml water was added thereto. The
solvent was then removed under reduced pressure.
The reaction product was dissolved into 5.0 ml of chloroform and the
solution was led to a column charged with silica gel, which was eluted
with a mixture of n-hexane and ethyl acetate (a mixing ratio of 20:1). The
elute was recrystalized with methyl alcohol to obtain 1.23 g (2.08 m
moles) of colorless, needle-like crystals, that is
transferulylstigmastanol, in 83% yield.
EXAMPLE 7
(1) Acetylation
Acetylation was carried out in the same manner as in Example 6.
(2) Formation of Acid Chloride
Acid chloride was formed in the same manner as in Example 6.
(3) Reaction with Phytostanol
The acid chloride was dissolved into 10 ml pyridine anhydride and the
resulting solution was dropped into 10 ml of a separately prepared
pyridine solution containing 1.72 g (4.2 m moles) campestanol, in an
ice-cooled condition. The solution was concentrated and then led to a
silica gel column which was eluted with a mixture of n-hexane and ethyl
acetate (a mixing ratio of 20:1), thus obtaining 1.45 g of a reactant.
(4) Deacetylation and Purification
1.4 g (2.25 m moles) of the reactant was dissolved into 42 ml of a liquid
mixture of chloroform and methyl alcohol (a ratio of 1:1), and to the
solution was added little by little sodium borohydride (Wako Jun'yaku K.
K.) in a total quantity of 478 mg (12.6 m moles). The reaction mixture was
allowed to stand at room temperature for 1.5 hours after the foaming had
ceased. The excessive sodium borohydride was inactivated by adding water
and then the solvent was evaporated under reduced pressure.
The resulting residue was dissolved into 4.5 ml chloroform and the solution
was subjected to a silica gel column which was eluted with a mixture of
n-hexane and ethyl acetate (a mixing ratio of 20:1) and recrystalized with
methyl alcohol. Thus, trans-ferulylcampestanol in a quantity of 1.15 g
(1.73 m moles) was obtained in 77% yield.
Although this invention has been described in conjunction with specific
exemplifying tests and preferred embodiments thereof, it is to be
understood that many variations may be made without departing from the
spirits and scopes thereof as defined in the appended claims.
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